이 작품은 미국 심장 협회 (박사 Beiyan 저우에 BGIA 7,850,037)에 의해 지원되었다.

1. 골수 세포의 분리 <ol><li> 6-8주 오래된 마우스에서 대퇴골과 경골 뼈를 분리, 머리를 헹구어 낸 후 뼈를 자르면. </li><li> 차가운 PBS 2 %의 열 비활성화 태아 소 혈청 (FBS) (3-5 ML / 마우스)에 골수를 플러시 21G 바늘과 10 ML의 주사기를 사용합니다. </li><li> 세포를 해리 21G 바늘을 통해 4-6 번 골수를 전달합니다. </li><li> 세포 덩어리, 뼈, 머리 및 다른 세포 / 조직을 제거하기 위해 70 μm의 셀 스트레이너를 통해 세포를 전달합니다. </li><li> NH 4 염소 용액 (0.8 % NH 4 망할 CIA 솔루션 스템 셀 기술) 3 볼륨을 추가하고, 적혈구를 제거하는 10 분 동안 얼음에 품어. </li><li> 4 ° C.에서 5 분 500 XG에서 세포를 스핀 다운 </li><li> 차가운 PBS 2 % FBS (20 ~ 50 ml의 세포의 양에 따라)에있는 세포 펠렛을 resuspend. </li></ol> 2. 유도 BMDM 형성 <ol><li> BMDM 성장 배지에서 분리 된 골수 세포 (2 × 10 6 세포 / M resuspend을L). </li></ol> BMDM 성장 매체 : Iscove의 수정 된 Dulbecco의 중간 (IMDM) + 10 % FBS + 15 % 여과 (0.2 μm의) L-929 세포 (ATCC, CCL-1) 문화 상층 액 (단핵구 콜로니 자극 인자, M-CSF를 포함) 또는 10 NG / ML M -CSF. 참고 : L-929 세포의 상층 액은 M-CSF 18이 포함되어 있습니다. 에어컨 중간, 5의 효과적인 활동을 보장하기 위해 X 10 5 L-929 세포 6-7일에 대한 T75 cm 2 플라스크에 씨앗을 품고 있으며, 에어컨 매체를 사용하기 전에 필터 0.45 μm의를 통해 수집하고 전달됩니다. 중간 분주은 1-2 개월 동안 즉시 사용할 또는 -80 ° C에 저장할 수 있습니다. <ol start="2"><li> 6 12 잘 조직 배양 플레이트 (실험 설계에 따라 다름) (코닝 공연자)의 씨앗 세포. </li><li> 3 일 신선한 BMDM 성장 매체를 변경합니다. </li><li> 하루에 7, 성숙 BMDM의 형성은 유동 세포 계측법 분석을 사용하여 평가됩니다및 형광 결합 항체는 CD11b 및 F4/80을 표현하는 세포를 감지합니다. </li></ol> 3. BMDM 편광 활성화 <ol><li> 7 일째에 신선한 자극을 매체로 변경 M1 활성화, IMDM 50 NG / ML IFNγ와 10 % FBS, 100 NG / ML LPS 또는 100 NG / ML LPS를 포함한 사용, M2 활성화와 10 % FBS를 포함 IMDM을 사용 10 NG / ML IL-4 및 / 또는 10 NG / ML IL-13. </li><li> 따뜻한 0.05 % 트립신을 사용하여 요리에서 그들을 분리에 의해 자극 BMDMs를 수집 PBS 10 % FBS를 포함한 두 번 세포를 세척 한 다음. </li></ol> 참고 : 차별화 후 성숙 식세포, 0.05 % 트립신 용액 (0.48 mM의 EDTA (에틸렌 다이아 민 테트라 초산), Invitrogen의를 포함) 또는 칼슘과 마그네슘이없는 PBS 또는 행크의 균형 버퍼 (HBSS)에 2-5 밀리미터 EDTA (에틸렌 다이아 민 테트라 초산)를 분리하고 resuspend을 위해 사용할 수 있습니다. 트립신을 포함하는 소화 매체를 사용하는 경우, 셀 쉬르의 손실을 방지하기 위해보다 10 분 동안 37 ° C에서 처리됩니다얼굴에 소화에 의한 단백질. <ol start="3"><li> CD11b, F4/80, CD11c, CD206, CD69, CD80 또는 표준 유동 세포 계측법 염색 프로 시저를 사용하여 다양한 시간 지점에서 CD86를 포함하여, 세포 표면 항원의 발현을 검출하기 위하여 항체를 사용합니다. </li><li> QRT-PCR을 이용하여 IL-1β, TNF-α와 IL-6 (M1 활성화) 또는 IL-10, IL-13, arginase1 및 PPARγ (M2 활성화) 등의 활성화 M1 및 M2 대식 세포의 특징적인 유전자의 발현을 확인합니다. 웨스턴 블롯 분석에 의한 M1 또는 M2 대식 세포의 활성화에 관련된 세포 신호 전달 경로의 활성화를 결정합니다. </li></ol>

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관심 없음 충돌 선언하지 않습니다.

우리는 여기에서 골수 전구 세포에서 유래 대 식세포의 활성화를 유도 할 수있는 간단하고 체외에서 쉽게 적응할 수있는 절차를보고합니다. 이 절차는 대식 세포의 편광에 대한 책임 메커니즘의 연구에 사용할 수 있습니다. 성숙한 대식 세포의 순도는이 프로토콜의 평균에게 95-99%을 사용하여 얻은하고 추가 정제 과정이 필요하지 않습니다. 대식 세포 분극, 자궁외 식 또는 유전자의 특정 최저의 맥락에서 이해 특정 유전자의 기능을 조사하려면 하루 7 세포의 다음과 같은 형질을 수행 할 수 있습니다. 이 프로토콜은 또한 형성, 성숙과 대식 세포의 표현형에 영향을 미치는 특정 요인이나 유전자의 영향을 조사하기 위해 7 일 문화의 창을 제공합니다. 활성화 된 대 식세포는 다양한 자극 3, 9, 19에 대한 응답으로 좋은 가소성을 가진 복잡한 세포 및 분자 프로파일을 표시합니다. 용그러나, 활성 프로파일이 완전히 중복되지 않는 예, LPS는 IFNγ 또는 종양 괴사 인자 (TNF)와 IL-4와 IL-13 M2 활성화를 자극 모두의 존재 더욱 강화 될 수있다 강력한 프로 염증성 M1 반응을 이끌어 3, 4, 9, 15, 19, 20. 이 프로토콜에 제시된 결과는 유동 세포 계측법, 정량적 RT-PCR과 웨스턴 블롯으로 분석 골수 유래 대 식세포와 실험의 일반적인 결과를 나타냅니다. 대 식세포의 활성화와 관련된 표면 항원 프레 젠 테이션 및 세포 신호 경로는 같은 대식 세포 분극 1-8 광범위한 문헌에 언급 달라집니다.

고전적인 염증 반응, 조직을 침투 대식 세포의 구별은 종종 호스트 조직 생리 기능 1-8 조절에 중요한 역할을 편광 정품 인증 상태를 표시합니다. 자극에, 대식 세포 활성화는 고전 (M1) 및 대체 (M2) 활성화 2, 4, 9로 정렬 할 수 있습니다. M1 대 식세포의 활성화는 TNF-α와 같은 염증성 사이토 카인의 생산에 이르는 수신자 같은 수용체 (TLR은) 핵 인자 카파 B (NFκB) / C 6 월 N-말단 키나제 1 (JNK1)의 활성화에 따라 IL- iNOS의의 1β 및 활성화하는 등의 질화물 산화물 (NO) 10, 11와 같은 활성 산소 종의 생산 증가의 결과. 반면, M2 대식 세포 활성화 모집 PPARγ, PPARδ, 또는 IL-4-STAT6 경로는 대체 만노오스 수용체 CD206의 상향 조절과 연관되어, 항 염증 (M2) 활성화 및 arginase 1 (이며 Arg1) 6, 12로 이어지는 - 14 &lt;/&gt;을 먹다. 골수 유래 대 식세포 (BMDM)는 활성화 된 대 식세포 15 편광을 제어하는 메커니즘을 이해하는 체외 모델의 이상을 제시한다. M2 대식 세포의 편광은 IL-4 및 / 또는 IL-13에 의해 유도 될 수 있지만, 특히 M1 대 식세포의 활성화는 리포 다당류 (LPS) 자극에 의​​해 유도 될 수있다. 성숙한 골수 유래 대 식세포 활성화 된 대 식세포는 CD11b, F4/80, CD11c, CD206, CD69, CD80 및 CD86 9, 16, 17 등의 표면 항원의 발현 유동 세포 계측법 분석을 통해 식별 할 수 있습니다. 또한, 시토 킨 생산 및 대식 세포 분극과 관련된 세포 신호 전달 경로의 변화는 정량적 RT-PCR 및 웨스턴 각각 내듯 측정 할 수 있습니다. 요약하면, 마우스 골수 유래 대 식세포는 체외에서 대식 세포 분극을 연구하는 중요한 모델이 될 수 있습니다.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>IMDM</td>
 <td>Thermo Scientific</td>
 <td>SH30259.01</td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal bovine serum</td>
 <td>Invitrogen</td>
 <td>10438-026</td>
 <td></td>
 </tr>
 <tr>
 <td>Murine GM-CSF</td>
 <td>PeproTech</td>
 <td> 315-03</td>
 <td></td>
 </tr>
 <tr>
 <td>NH4Cl</td>
 <td>StemCell Technologies</td>
 <td>7850</td>
 <td></td>
 </tr>
 <tr>
 <td>L-929</td>
 <td>ATCC</td>
 <td>CCL-1</td>
 <td></td>
 </tr>
 <tr>
 <td>70 &mu;m cell strainer</td>
 <td>BD Biosciences</td>
 <td>352350</td>
 <td></td>
 </tr>
 <tr>
 <td>10 x PBS</td>
 <td>Thermo Scientific</td>
 <td>AP-9009-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-mouse CD11b-APC</td>
 <td>eBioscience</td>
 <td>17-0112-81</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-mouse F4/80-FITC</td>
 <td>eBioscience</td>
 <td>11-4801-81</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-mouse CD69-PE</td>
 <td>eBioscience</td>
 <td>12-0691-81</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-mouse CD86-PE</td>
 <td>eBioscience</td>
 <td>12-0862-81</td>
 <td></td>
 </tr>
 <tr>
 <td>Propidium Iodine</td>
 <td>Invitrogen</td>
 <td>P3566</td>
 <td></td>
 </tr>
 </tbody>
</table>

investigation of macrophage polarization using bone marrow derived macrophages

이 문서는 대식 세포 분극을 조사하기 위해 체외 모델에서 쉽게 쉽게 적응을 설명합니다. GM-CSF/M-CSF의 면전에서, 골수에서 조혈 줄기 / 전구 세포 M1 또는 M2 자극 한 다음, 단핵구 분화에 연결됩니다. 활성 상태는 세포 표면 항원 유전자 발현 및 세포 신호 전달 경로의 변화에​​ 의해 추적 할 수 있습니다.

BMDM 생성 과정의 개략적 인 설명은 (그림 1) 표시됩니다. 그들은 CD11b + F4/80 + 세포의 95-99% (그림 2)를 나타냅니다 때 성숙한 대식 세포의 순도는 7 일에 관찰 할 수 있습니다. 편광 식세포가 CD11b, F4/80에 대한 항체를 사용하여 검사 할 수 있습니다, CD11c와 CD206는 유동 세포 계측법 분석에 의해 따랐다. 그림 3에서와 같이, M1 대 식세포는 다음과 같이 발견 된 CD11b + F4/80 + CD11c + CD206-세포 (Q2), M2 대식 세포는 CD11b 반면 + F4/80 + CD11c-CD206 + 세포 (Q4). BMDM 활성화 상태가 증가 세포의 크기 (그림 4A, FSC-A에서 오른쪽으로 이동) 및 표면 항원의 증가 풍부한 CD69, CD80, 또는 대식 세포에서 CD86에 의해 확인 될 수있다 (문 : CD11b + F4/80 +) 그림에서와 같이 4B. 시토 킨 생산, 유전자 발현 및 M1 또는 M2 대식 세포의 세포 신호 전달 경로의 활성화는 정량적 RT-PCR이나 웨스턴 블로 팅을 사용하여 평가할 수 있습니다.의 그림 5와 같이 arginase 1 (이며 Arg1)와 PPARγ 수준은 10 NG / ML IL-4 자극에 M2 대식 세포에서 증가 하였다, IL-1β와 TNFα 생산이 100 NG / ML LPS로 자극 식세포 (M1) 증가 하였다 반면 , 어떤은 P65 활성화 (그림 6)에 의해 동반됩니다. <img src="/files/ftp_upload/50323/50323fig1.jpg" alt="그림 1" fo:content-width="5.5in"/> 그림 1. 격리, 형성 및 마우스 BMDMs의 자극에 대한 계획. 단계 절차에 의해 단계는 텍스트에 설명되어 있습니다. 간단한, 대퇴골 및 경골 뼈 PBS가 2 % 열 불 활성화 FBS와 보충하여 플러시 6~8주 마우스 골수 세포에서 수집됩니다. 적혈구는 NH4Cl 용액으로 용해 된 후, 세포는 세포 인구의 순도 성숙과 분석에 의해 다음 칠일에 대한 BMDM 성장 배지에서 배양된다. 대식 포의 분석<html> larization, 세포 M2 활성화를 위해 + IFNγ M1, 또는 IL-4 및 / 또는 IL-13 LPS 또는 LPS로 자극 하였다. 극화 된 대 식세포는 세포 형태의 변화, 표면 마커 프레 젠 테이션, 시토 킨 생산 및 활성화 세포 신호 전달 경로의 기준으로 평가할 수 있습니다. <img src="/files/ftp_upload/50323/50323fig2.jpg" alt="그림 2" fo:content-width="6in" fo:src="/files/ftp_upload/50323/50323fig2highres.jpg"/> 그림 2. BMDM 형성 유동 세포 계측법 분석. (A) BMDMs 파편과 복합체를 제거하는 FSC와 SSC에 처음 문이었다. (B) 성숙한 BMDMs는 다음과 같이 정의 된 CD11b의 백분율로 표시되는 순도 + F4/80 + 모집단 (오른쪽) FSC / SSC에 문이 부모의 인구. s/ftp_upload/50323/50323fig3highres.jpg &quot;/&gt; 그림 3. . 대식 세포 편광 분석 조직 침투 대식 세포는 CD11b + F4/80 + 세포로 정의되며, M1 대 식세포는 CD11b 있습니다 + F4/80 + CD11c + CD206-세포, M2 대식 세포는 CD11b + F4/80 + CD11c-CD206 + 세포 수 있습니다 반면. <img src="/files/ftp_upload/50323/50323fig4.jpg" alt="그림 4" fo:content-width="6in" fo:src="/files/ftp_upload/50323/50323fig4highres.jpg"/> 그림 4. 유동 세포 계측법에 의한 대 식세포의 활성 분석. (A) 활성화되면, 대식 세포의 크기는 M0 (치료) 대 식세포에 비해 자극 후 24 시간에서 M1 또는 M2 대식 세포의 FSC-A의 변화에 의해 입증 증가한다. (B) 활성화 관련 표면 마커는 IL4이나 LPS 자극 후 유동 세포 계측법에 의해 분석 하였다. 초기 응답 마커 CD69는 5 또는 24 시간 후 자극에서 평가되었다 CD80 및 CD86 마르크RS는 자극 후 48 시간에서 분석 하였다. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50323/50323fig4large.jpg" target="_blank">큰 그림을 보려면 여기를 클릭하십시오</a> . <img src="/files/ftp_upload/50323/50323fig5.jpg" alt="그림 5"/> 그림 5. 대식 세포의 대안 활성화. 24 시간 동안 10 NG / ML IL-4로 자극 식세포를 수집하고 총 RNA가 추출되었다. 정량적 RT-PCR 분석을 사용하여 치료 대식 세포에 비해 높은 arginase 1 (이며 Arg1)와 PPARγ 발현 M2 대식 세포에서 발견되었다. <img src="/files/ftp_upload/50323/50323fig6.jpg" alt="그림 6" fo:content-width="5.5in" fo:src="/files/ftp_upload/50323/50323fig6highres.jpg"/> 그림 6. 대 식세포의 고전 활성화. (A) 총 RNA 100 NG / ML L로 자극 대식 세포에서 추출 된24 시간 정량적 RT-PCR 분석에 사용을위한 PS. IL-1β와 TNFα 등의 염증성 사이토 카인의 발현은 M1 대식 세포에서 증가했다. P65에 대한 항체를 사용하여 결정하고 서양 블롯 분석에서 P65을 인산화로 (B) NFκB 경로의 활성화는 LPS에 의해 유도되었다.

Watch this Scientific Journal Video about Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages at JoVE.com

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

이 문서는 쉽게 쉽게 적응을 설명<em&gt; 체외에서</em대식 세포 분극을 조사하기&gt; 모델. GM-CSF/M-CSF의 면전에서, 골수에서 조혈 줄기 / 전구 세포 M1 또는 M2 자극 한 다음, 단핵구 분화에 연결됩니다. 활성 상태는 세포 표면 항원 유전자 발현 및 세포 신호 전달 경로의 변화에​​ 의해 추적 할 수 있습니다.

골수 유래 대 식세포를 사용하여 대식 세포 분극 조사

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

investigation-macrophage-polarization-using-bone-marrow-derived

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

65.9K 조회수.  Texas A&M University. 이 문서는 쉽게 쉽게 적응을 설명 체외에서 모델. GM-CSF/M-CSF의 면전에서, 골수에서 조혈 줄기 / 전구 세포 M1 또는 M2 자극 한 다음, 단핵구 분화에 연결됩니다. 활성 상태는 세포 표면 항원 유전자 발현 및 세포 신호 전달 경로의 변화에​​ 의해 추적 할 수 있습니다.

비디오: Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO2 starvation, using medium with high a pH, or the addition of interferon gamma (IFN&gamma;). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO2 and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFN&gamma; and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii to bradyzoites using a high pH growth medium with low CO2 and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFN&gamma; and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii converts to a cyst form in response to environmental stresses, which can be mimicked in tissue culture models. This video demonstrates techniques to examine cyst wall formation by activating bone marrow-derived macrophages or changing growth medium pH in fibroblast cells.

활성 골수 - 유래 Macrophages과 Bradyzoite Toxoplasma gondii 약관의 낭종의 벽 형성

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii ...

연구

면역학 및 감염병학

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

추적 연구 Qdot Nanocrystals와 Murine 골수 파생 돌기 세포의 생성 및 라벨링

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

뼈 대식 세포 생산에게 골수 유래

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

분리 및 설치류 골수의 정맥 주사 유래 단핵구

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage&#8217;s metabolism and phenotype.

실시간 세포 외 유출 분석을 사용하여 편광 M1과 M2 골수 유래 대 식세포의 대사 특성

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.

Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space.

쥐 뒷다리 긴 뼈 해부 및 골수 격리

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

마우스 골수에서 생성 및 GM-CSF의 식별 파생 폐포와 같은 대 식세포와 수지상 세포

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

대 식세포 콜로니 자극 인자 차별화 된 인간 단핵구 유래 대 식세포의 문화

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Murine 골 복 막 대 식 세포 활성화에 대 한 항 체 기반 약물 효과의 평가

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.